Biochemical and Cellular Characterization of Helicobacter pylori RecA, a Protein with High-Level Constitutive Expression

Abstract

Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4,6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system.

Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005

“…For H. pylori this has indeed been shown and suggested as explanation for the absence of recA induction (Orillard et al. ). Thus, this could also be a hypothesis to test for.…”

“…() and H. pylori following UV and gamma radiation (Orillard et al. ). RecA is crucial for DNA repair and essential photosynthetic prokaryotes (Owttrim and Coleman ), thus probably also active in Arthrospira sp PCC8005 after irradiation.…”

Following transforming DNA in Helicobacter pylori from uptake to expression

“…It was first necessary to set up the conditions for normal growth during image acquisition. Figure A and Supporting Information Movie S1 present examples of time‐lapse microscopy of an H. pylori 26695 overnight culture, showing that it is possible to follow the dividing bacteria for over 12 h. From several similar experiments we determined a 2.5 h generation time under conditions used for image acquisition, in good agreement with the generation time observed for the same strain in liquid cultures (Orillard et al ., ).…”

Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori

“…Moreover, multiple DNA repair pathways contribute to efficient colonization 20 even while the surrounding host tissue accumulates DNA lesions 21, 22 . Recent work has shown that H. pylori strains constitutively express DNA repair proteins such as RecA and thus lack a classic SOS response to DNA damage 23, 24 . Upon DNA damage, H. pylori instead upregulates natural competence which promotes chronic persistence, likely through enhanced genetic diversification 23, 25 .…”